Are muscles and gonads from lipophilic or paralytic phycotoxin contaminated Pectinidae always safe?

Transcription

1 Are muscles and gonads from lipophilic or paralytic phycotoxin contaminated Pectinidae always safe? Frémy Jean Marc 1*, Arnich Nathalie 1 and Biré Ronel 2 1 AFSSA (Agence Française de Sécurité Sanitaire des Aliments), Direction de l Evaluation des Risques Nutritionnels et Sanitaires, avenue du Général Leclerc, Maisons-Alfort, FRANCE. * corresponding author: jeanmarc.fremy@anses.fr 2 AFSSA (Agence Française de Sécurité Sanitaire des Aliments), Laboratoire d Etudes et de Recherches sur les Aliments et leurs Process, 23 avenue du Général De Gaulle, Maisons-Alfort, FRANCE. Commission decision 2002/226/CE may authorise the harvesting of Pectinidae belonging to the species Pecten maximus and Pecten jacobaeus with a domoic content in whole body exceeding 20 mg/kg but lower than 250 mg/kg, if contaminated parts are removed. The French regulatory authorities asked AFSSA to estimate the relevance for extending the scope of this decision to other phycotoxins contamining these two species and to other species. And if yes, can freezing process have an influence on phycotoxins anatomical distribution? From several studies performed on the genus Pecten found in the litterature, it is observed that digestive gland contains the highest level of toxins, then mantle and gills. Regarding contents found in gonad and adductor muscle, the concentrations are generally low and very low, respectively. However, compiled data from different studies performed on paralytic toxins contents in Pectinidae show that any constant correlation between tissues could not be established. Specific data regarding the both species Pecten maximus and Pecten jacobaeus are too limited to set a range of contents in the view of extend the scope of the Commission decision for lipophilic or paralytic phycotoxins. Moreover, some experimental studies on paralytic toxins have shown that during the detoxification period, kidneys can become highly toxic. And this particular organ can stay linked to the adductor muscle during the removal process from the whole body. Regarding the influence of freezing process, data from scallops Patinopecten yessoensis show a possible post mortem transfer or exchange from gonad to muscle. Are discussed also the effects of freezing/thawing on the integrity of shellfish tissues andon the stability of phycotoxins. Keywords: Pectinidae, phycotoxins, contamination, edible parts, safety 1

2 Introduction Like all shellfish and especially filter-feeders, Pectinidae are sensitive to contamination by phycotoxins (toxins produced by marine phytoplankton). In France, Pectinidae production essentially consists of harvest in natural beds mainly situated off the coast and includes mainly two categories: King scallops ( Coquilles Saint Jacques in French).and queen scallops ( pétoncles in French). Over the last few years, algal toxin contamination of shellfish has been observed, thanks to the monitoring of both production areas and marketed products. Regulation (EC) No. 854/2004 of 29 April 2004 lays down specific rules for the organisation of official controls on products of animal origin intended for human consumption and therefore applies to the presence of phycotoxins in shellfish. Regulation (EC) No. 853/2004 of 29 April 2004 sets permitted limits for amnesic shellfish poison (ASP), paralytic shellfish poison (PSP) and lipophilic toxins (okadaic acid, dinophysistoxins, pectenotoxins, yessotoxins and azaspiracids) above which shellfish are considered unfit for human consumption. This regulation offers the possibility of placing on the market only the edible parts of the shellfish, which have been proven not to exceed the sanitary limits (whereas the analyses of the whole meat may exceed them). Following recurrent episodes of contamination of bivalve mollusc beds in Scotland by ASP toxins, the Commission adopted Decision 2002/226/EC, authorising the sale in certain conditions of 2 species of King scallops (Pecten maximus and Pecten jacobaeus) when the phycotoxins in the whole meat exceed the permitted limits. The products intended for consumption then consist of just the adductor muscles and/or the gonads. The parts where the toxins are concentrated must be removed (digestive gland or hepatopancreas - and mantle). In these conditions the harvest permit is accompanied by very strict control measures: - the level of domoic acid in the shellfish must be lower than 250 mg/kg in the whole meat and lower than 4.6 mg/kg in the parts intended for sale under the conditions of this decision; - the channelling of the products must be strictly supervised by the authorities responsible for fishing and placing on the market; - each batch of final product must be analysed in order to prove that it is fit for human consumption (the level must be less than 20 mg/kg). This decision was based on scientific data and has not yet been extended to either other species of Pectinidae or other phycotoxins. The French Food Safety Agency (AFSSA) was requested by the French management authorithies for scientific and technical opinion on the control of the phycotoxin risk in Pectinidae by the introduction of an evisceration process. Three main questions were asked: 1. Would it be possible to extend the system set up by Decision 2002/226/CE for 2 species of King scallops contaminated by ASP toxins to the other families of toxins (lipophilic or PSP toxins), in order to place on the market products fit for consumption (eviscerated products), in spite of the contamination of the product (whole meat) in the production area? In particular, is it necessary to introduce a limit at harvest similar to the 4.6 mg/kg limit on domoic acid for ASP toxins in the parts intended for sale? 2. Would it be possible to extend these measures (lipophilic, ASP, PSP toxins) to all Pectinidae produced in France and in particular to queen scallops subject to the technical conditions allowing the routine removal of the hepatopancreas? 2

3 3. Can the freezing process of shellfish harvested in the expectation of their treatment by evisceration generates an extra risk (transfer of toxins into the muscle, bursting of the hepatopancreas during freezing )? The present paper summarizes available data in published litterature providing informations with regards to these questions and presents the key points of AFSSA s opinion on 14 novemeber Some recommendations are also suggested to complete the lack of data for some aspects. 1. Application of evisceration process to King scallops contaminated by lipophilic and/or PSP toxins First of all, it is necessary to emphasise the small number of studies available on the distribution of lipophilic and PSP toxins in the 2 species of scallops cited in the request Organ distribution of lipophilic toxins Brana Magdalena et al. (2003) reported that the digestive gland of Pecten maximus scallops concentrates a large part of azaspiracids (AZAs) (85%), but not all of it, as smaller concentrations of toxins are found in the other organs. An experimental contamination study of bay scallops (Argopecten irradians) using cultures of Prorocentrum lima which produce okadaic acid (OA), dinophysistoxin-1 (DTX-1) found the following distribution: digestive gland (76 %), gonad (12 % ), muscle (4 %), mantle (4 %), gills (4 %) (Bauder et al., 1996). A similar distribution of AZAs has been described in shellfishes other than the 2 types of scallops concerned by the request, namely mussels Mytilus edulis (Flanagan et al., 2000 ; James et al., 2002a, 2002b).Hess et al. (2005) also found that the concentration of AZAs is 5 times higher in the digestive gland than in the whole meat of mussels originating from Ireland and Norway. Blanco et al. (2007) demonstrated that all or nearly all okadaic acid is accumulated in the digestive gland of mussels Mytilus galloprovincialis Organ distribution of PSP toxins An experimental study conducted by Lassus et al. (1996) showed that when Pecten maximus scallops are exposed to a toxinogenic culture of Alexandrium tamarense, the PSP toxins accumulate mainly in the digestive gland, then to a lesser degree in the gonads and the adductor muscle (respectively, 2,620, 148 and less than 50 µg eq STX/100 g). In the other Pectinidae species, Patinopecten yessoensis and Placopecten magellanicus, the highest toxin concentrations were also found in the digestive gland (Lassus et al., 1996). A synthetic review conducted by Shumway and Cembella (1993) on Pecten genus species also concluded that the majority of the accumulation was in the digestive gland. These authors observed lower contents in gonads and adductor muscle but no arythmetic correlations could be made between toxicity levels in digestive gland and other tissues. After the end of the exposure period, depuration occurs, with a reduction in the toxin concentration in the digestive gland, the gonads and the adductor muscle, but an increase in the kidneys where the toxins may be biotransformed into more toxic metabolites belonging to the same family, still retained after 20 days (Lassus et al., 1996). These authors emphasise that the kidneys are often present in scallops sold "eviscerated" on the market. 3

4 In conclusion, regarding lipophilic and PSP toxins, the limited data available on the species Pecten maximus seem to indicate a toxin accumulation mainly in the digestive gland. Nevertheless, certain data on contamination by PSP toxins show that the kidneys may still be present after evisceration. And the kidneys retain toxic metabolites, even 3 weeks after the end of the exposure period. No data is currently available concerning the species Pecten jacobaeus. Consequently, it is not possible at present to establish a reliable correlation between the toxin levels in the digestive gland and those in the other body fractions (gonads, muscle and kidney), which would make it possible to determine, as was the case for domoic acid, specific conditions of removal and treatment (evisceration) for these shellfish, if the regulatory limits for lipophilic or PSP toxins are exceeded, thus allowing a satisfactory level of safety to be guaranteed for the consumer. 2. Application of evisceration process to other Pectinidae species Experimental data provided by IFREMER (Amzil, personal communication) regarding levels of lipophilic toxin (okadaic acid, dinophysistoxins and azaspiracids) in King scallops ( Coquille St Jacques in French but the name of species is not specified) collected off Roscoff in October and November 2006 show a preferential accumulation of these toxins in the digestive gland, with concentrations above the safe limit for azaspiracids. In the opposite, the toxin concentrations in the whole meat, the muscle and the other organs were considerably lower than regulatory limits. On the basis only of these data, it does not therefore seem to be necessary to eviscerate scallops, as long as the regulatory limits for the whole meat are not exceeded. However, it would be preferable to have more experimental data for lipophilic, PSP and ASP toxins in order to gain a more representative picture of the level of contamination of all species of scallops that could be harvested along the French coasts before to decide whether it is advisable to introduce an evisceration process. 3. Consequences of freezing process Several aspects must be considered: the effects of freezing/thawing on the physical integrity of the shellfish matrix; the stability of the phycotoxins on freezing; the effects of freezing on the free fatty acids and their impact on toxin detection Effects of freezing/thawing on the physical integrity of the shellfish matrix To our knowledge there are no bibliographic data regarding the impact of shellfish structure alteration during freezing and the migration of toxins from one tissue to another. However, it is possible that during freezing, the structure of tissues highly contaminated with toxins is altered and that consequently, the movement of water due to the melting of the crystals on thawing is liable to cause a migration of the toxins to neighbouring tissues. 4

5 The extent of this phenomenon will depend on the contamination levels and probably on the nature of the toxins (fat-soluble or water-soluble) as well as their "degree" of fixation in the matrix. Work to study these phenomena would be useful to obtain experimental data. However, during a routine control of PSP toxin occurrence on imported pacific scallops (Patinopecten yessoensis) into France, a study was conducted by Fremy et al. (1993) on a sampling of the batch of bags containing associated frozen muscles and gonads. Toxin content in associated muscles (M) and gonads (G) was tested separately by the AOAC mouse bioassay. The results were recorded as positive (+) or negative (-) according to being above or below the limit of sensitivity of the assay (320 µg equiv. STX per kg). Three cases of data were found: M-/G-, M-/G+, and M+/G+. Regarding the last case, in some samples, toxin contents were at the same levels for both M and G. Since some experimental observations showed that the toxin concentration is higher in gonads than that of in muscle (Lassus et al., 1996) two options could explain the phenomenon of equal toxin levels between some samples of associated muscle and gonad: (i) a post mortem transference from gonad to muscle before- or failure during- the freezing process or frozen storage reaching an equilibrium between the two organs; (ii) the presence of highly toxic kidneys which remained often attached to the muscles when animals were eviscerated as mentioned by Lassus et al.(1996) which would enhance the toxicity of tested samples of muscles being in fact samples of associated muscles and kidneys. Different studies have shown that the phenomenon of freezing is accompanied by dehydration of the surface of the tissues, a sublimation phenomenon which depends on the characteristics of the matrix, but also on the ambient conditions and which leads to mass loss in the matrix (Campaneone et al. 2001; Campaneone et al. 2005; Olguin et al. 2008). Water loss due to storage conditions was also observed by Smith et al. (2006) but at higher temperatures (between 5 and 12 C). Indeed, these authors report that for increasing temperatures, the water loss in Pecten maximus scallops during storage is accompanied by an increase in the domoic acid level. We may therefore venture the hypothesis that during storage at negative temperatures, water loss resulting from sublimation could lead to an increase in domoic acid in Pecten maximus scallops. Although this has not been tested for the other marine toxins, we can reasonably assume that the phenomenon will be similar; in any case it is worth checking.it is important to point out that if the dehydration of the tissues leads to an increase in the toxin concentration (but not the quantity), this may also have other consequences. Indeed, Smith et al. (2006) report that due to the water loss, the domoic acid is bound more strongly to the matrix and becomes less extractable, a phenomenon also observed by the Canadian National Research Council for certified reference materials for domoic acids stored for "a long time" (no information on the exact storage time). In terms of the health impact, this raises the question of the bioavailability of the toxin in the organism after storage, aspects which are largely unknown for marine toxins Effects of freezing on stability of toxins The bibliographic data relating to the stability of toxins when freezing scallops are quite limited. So, data obtained for shellfish other than Pectinidae; are also reported Domoic acid Leira et al. (1998) showed that part of the domoic acid contained in scallops (Pecten maximus) is lost in an amount that depends on the freezing time : the concentration of domoic acid in whole scallops decreased by 43% after 180 days of frozen storage, the 5

6 decrease being mostly in the hepatopancreas. Vale et al. (2002) observed a slight drop in the domoic acid level in a sample of Venerupis pallustra clam (of the order of 10%) after one month's storage for freezing temperatures of -15 and -40 C. No variation in the concentration of this toxin was reported by McCarron et al. (2007) for a reference material prepared from mussels Mytilus edulis and stored at -20 C for 240 days Lipophilic toxins The study carried out on a reference material prepared from mussels Mytilus edulis and stored at -20 C confirmed the stability of okadaic acid, dinophysistoxin-2 and azaspiracids-1 to 3 over 240 days (McCarron et al., 2007) PSP toxins There is no significant change in any toxin type such as C toxins (C1-2), GTX (gonyautoxins) 1-4, STX (saxitoxin) and NEO (neosaxitoxin) in scallop digestive gland homogenates and a mixture of purified PSP toxins (B1, C1-2, GTX 1-4, STX and NEO) during 1 year storage at 35 C and at different ph levels (Indrasena and Gill, 2000) Effects of freezing on free fatty acids and their impact on toxin detection. Fukushi et al. (2003) reported that the storage at -20 and -45 C for 30 days of Patinopecten yessoensis scallops is accompanied by an increase in the free fatty acid (FFA) content, due to enzymatic hydrolysis of the triglycerides. The FFA content is higher at -20 than at -45 C and increases with storage time. Although this variation in the FFA content in scallops has no impact in itself on human health, some studies have shown that FFAs are likely to interfere during the mouse bioassay used as a reference method for lipophilic toxins (Takagi et al., 1984; Lawrence et al., 1994; Suzuki et al., 1996). This phenomenon can generate false positives in the mouse bioassay and lead to the withdrawal of products that do not in fact represent a health risk for the consumer. Conclusion Regarding lipophilic and PSP toxins, the available data on the species Pecten maximus seem to indicate a toxin accumulation mainly in the digestive gland. This preferential accumulation could envisage the principle of an evisceration process as is already the case for domoic acid (Commission Decision 2002/226/CE of 15 March 2002). However, it is not possible at present to establish a reliable correlation between the toxin levels in the digestive gland and those in the other body fractions (gonads, muscle and kidneys). Moreover, certain data on contamination by PSP toxins show that the kidneys may still be present after evisceration, and the kidneys retain toxic metabolites, even 3 weeks after the end of the exposure period. In addition, there are no data available regarding the Pecten jacobaeus species, either for lipophilic or for PSP toxins. Consequently, it is not possible at the present time to determine conditions of removal and treatment (evisceration) for these 2 species of scallops if the regulatory limits for lipophilic or PSP toxins are exceeded. Studies are required on the decontamination by evisceration of the species Pecten maximus and Pecten jacobaeus contaminated by these toxins, in order to identify harvesting limits which would guarantee the sanitary safety of consumers. The extension of this evisceration process approach to other Pectinidae and in particular to the queen scallops sold as "pétoncles" in France requires, on the one hand, that control 6

7 authorities issue an official list of species of Pectinidae produced and sold in France under the names "coquilles Saint-Jacques" (as King scallops ) and "pétoncles" (as queen scallops ); and on the other hand, that studies on decontamination by evisceration be carried out on these species. The freezing/thawing of shellfish before evisceration is liable to lead to an extra risk related in particular to the alteration of the contaminated tissues, facilitating the crosscontamination of other organs. Furthermore, if the ambient conditions are not stable, this process might enhance the phenomenon of sublimation with water loss and therefore an increase in the toxin concentration. To avoid this phenomenon, it is necessary to ensure that the freezing step is rapid and there is no break in the cold chain. Consequently, it will be necessary to conduct studies intended to determine the effects of freezing and thawing on the phycotoxin level and their impact in terms of toxin detection (effects on free fatty acids). In fact, as long as the reference method remains the mouse bioassay, the increase in the free fatty acid content during cold storage of shellfish can generate false positives. References Bauder A. G., Cembella A. D., Quilliam M. A., (1996). Dynamics of diarrhetic shellfish toxins from the dinoflagellate, Prorocentrum lima, in the bay scallop, Argopecten irradians. In : Harmful and Algal Blooms. Ed. Oshima T. and Fukuyo Y. Intergovernmental Oceanographic Commission of UNESCO, pp Blanco, J., Marino, C., Martin, H., & Acosta, C. P. (2007). Anatomical Distribution of Diarrhetic Shellfish Poisoning (DSP) Toxins in the Mussel Mytilus galloprovincialis. Toxicon, 50(8). Brana Magdalena A., Lehane M., Moroney C., Furey A., James K.J. (2003). Food safety implications of the distribution of azaspiracids in the tissue compartments of scallops (Pecten maximus). Food Addit Contam 20, Campaneone L.A., Salvadori V.O., Mascheroni R.H. (2001). Weight loss during freezing and storage of unpackaged foods. Journal of Food Engineering 47, Campaneone L.A., Salvadori V.O., Mascheroni R.H. (2005). Food freezing with simultaneous surface dehydration: approximate prediction of weight loss during freezing and storage. International Journal of Heat and Mass Transfer 48, Flanagan A. F., Donlon J., Kane M. (2000). Localisation of azaspiracid, a recently discovered shellfish toxin, within the blue mussel Mytilus edulis. In Ninth International Conference on Harmful Algal Blooms. Ed. G. Hallegraeff, C. J. Bolch, S. I. Blackburn & R. J. Lewis. Hobart, Tasmania: Intergovernmental Oceanographic Commission of UNESCO. Fremy J.M., Ledoux M., Bilodeau M., MajorM., Murail I., Jamet J. (1993) Variation de la contaminantion par des phycotoxines paralysantes de coquilles St Jacques importées d Asie. Sciences des Aliments 13, Fukushi A., Imamura T., Takahashi H., Itabashi Y., Suzuki T. (2003). Increase of free fatty acids in the hepatopancreas of scallops kept in freezer. Fisheries Science 69 (5), Hess, P. Nguyen L., Aasen J., Keogh M., Kilcoyne J., McCarron P., Aune T (2005). Tissue distribution, effects of cooking and parameters affecting the extraction of azaspiracids from mussels, Mytilus edulis, prior to analysis by liquid chromatography coupled to mass spectrometry. Toxicon 46 (1), Indrasena W.M. and Gill T.A. (2000). Storage stability of paralytic shellfish poisoning toxins. Food Chemistry, 71, pp James K.J., Lehane M., Moroney C., Fernandez-Puente P., Satake M., Yasumoto T., Furey A. (2002a). Azaspiracid shellfish poisoning: unusual toxin dynamics in shellfish and the increased risk of acute human intoxications. Food Addit Contam 19, James K. J., Furey A., Lehane M., Ramstad H., Aune T., Hovgaard P., Morris S., Higman W., Satake M., Yasumoto T. (2002b). First evidence of an extensive northern European distribution of azaspiracid poisoning (AZP) toxins in shellfish. Toxicon 40,

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